Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease affecting skin and mucous membranes that is mediated by circulating antibodies against a keratinocyte desmosomal cadherin, desmoglein (Dsg) 3, which mediates cell adhesion. Recent studies using phage display indicate the presence of both pathogenic and non-pathogenic monoclonal autoantibodies (mAbs) in PV patient sera. The pathogenic antibodies directly cause blister formation by interfering with cell adhesion. Although current immunosuppressive regimens targeting the total antibody pool have led to reduced patient mortality, morbidity associated with the side effects of treatment is significant and underscores the need for therapies that specifically target pathogenic anti-Dsg antibodies. These observations suggest a novel approach to treatment would be to use small molecule reagents that block the binding of pathogenic mAbs to Dsg. The target has been established with the extracellular portion of Dsg3 displayed on protein G beads and the pathogenic monoclonal antibody expressed as a single chain variable fragment antibody (scFv)-enhanced green fluorescent protein (EGFP) construct. A homogeneous flow cytometry assay has been implemented in 384-well plate format and an initial screen of the Prestwick Chemical Library has been completed. The assay routinely performs with Z' values in the range of 0.5 - 0.8. We will use this assay to screen the Molecular Libraries Small Molecule Repository (MLSMR) to identify molecules that interfere with the binding of pathogenic scFv anti-Dsg3 to disease important epitopes of Dsg3. Secondary assays based on the flow cytometry assay will be used to validate active compounds identified in the primary screen. Identified compounds will be tested for inhibition of pathogenicity of pemphigus antibodies in tertiary follow-up biologic assays of human skin organ culture. These systems are well established in the PI's laboratory. This proposal is expected to result in novel lead compounds with the potential of revolutionizing the treatment of pemphigus. PUBLIC HEALTH RELEVANCE: Pemphigus vulgaris is a disfiguring and potentially fatal blistering autoimmune disease. Current therapy is to suppress the immune system, which results in many potential adverse effects. This proposal seeks therapy directly targeted only to the autoantibodies that actually cause the blisters in this disease.